There is considerable interest in developing techniques for determining the presence of analytes of biological origin in samples, particularly clinical samples. One technique uses the complementary binding known as hybridization that takes place between complementary strands of nucleic acids such as DNA and RNA to identify the presence of analytes containing DNA or RNA in samples.
Specific hybridization techniques have been developed for determining the presence of a specific virus, bacterium, or other organism in a biological sample, as well as for detecting genetic defects in mammalian cells. Among the recently developed techniques are those that rely on the formation of a covalent bond between the target and the reagent polynucleotide strands. In one such technique, a nucleic acid reagent (probe) is created containing a covalently linked, photoactivatable moiety that is capable of forming covalent bonds with the analyte upon photoactivation. If a probe and analyte are mixed under hybridizing conditions and the linking group is photoactivated, covalent bonds are formed that bind the two strands together. If the probe also contains a detectable signal, rigorous techniques for separating single and double-stranded nucleic acids can be utilized to determine the presence of analyte in the sample by determining the presence of crosslinked nucleic acid strands. For example, U.S. Pat. No. 4,599,303 to Yabusaki et al. describes nucleic acid hybridization techniques that employ probes that are crosslinkable to target sequences.
These prior techniques have typically relied on the use of a photoactivatible group that is covalently attached to a base residue of a polynucleotide. However, synthesis of the known photoactivatible probes is difficult, particularly in large scale.
Accordingly, new techniques that rely on more readily available analogues of nucleotides that are photoactivatible are desirable.